Our long term goal is to understand the function of the protein antioxidant system that protects reactive protein sulfhydryl groups from oxidative damage, i.e, the protein S-thiolation/ dethiolation protection system. This application is a one year pilot project to investigate the role of protein S-thiolation/dethiolation in an aging animal model. We will I) determine whether aging causes increased protein S-thiolation in rats, identifying individual modified proteins by a newly proposed method, and whether irreversibly modified forms of carbonic anhydrase III are a consequence of inadequate protection by S-thiolation/dethiolation; 2) determine whether the dethiolation system, i.e., glutaredoxin and thioredoxin, is less effective as a consequence of aging; and 3) develop a method to produce an antibody to glutathione that can be used to detect protein-bound glutathione, i.e., S-glutathiolated proteins, in future experiments. These experiments are our first attempt to relate the function of the protein antioxidant system (S-thiolation/dethiolation) to altered signal transduction, and gene transcription in an aging animal model. The proposal is based on a recent report of increased S-glutathiolation of carbonic anhydrase III in the liver of older rats. This unique result suggests that the protein antioxidant system is less effective in older animals, potentially leading to irreversible modification (oxidation) of these proteins as well as increased S-thiolation of a number o important protein cysteines. Since we propose that the cysteines required for lipidation, ADP-ribosylation, cysteine- mediated proteolysis in apoptosis, and redox control of transcription are all protected by the S-thiolation/dethiolation antioxidant system, any loss of the protective role it performs would certainly be detrimental to proper cell functions in aged animals. The experiments describe methods to assess both protein S- thiolation and irreversible oxidation in older animals.